Proteases constitute a large and important group of enzymes involved in diverse physiological processes such as blood coagulation, inflammation, reproduction, fibrinolysis, and the immune response. Numerous disease states are caused by, and can be characterized by, the alterations in the activity of specific proteases and their inhibitors. The ability to measure these proteases in research or clinically is significant to the investigation, treatment and management of disease states. For example, caspases 3 and 7 are members of the cysteine aspartyl-specific protease (also known as the aspartate specific-cysteine protease, “ASCP”) family and play key effector roles in apoptosis in mammalian cells (Thomberry et al., 1992; Nicholson et al., 1995; Tewari et al., 1995; and Fernandes-Alnemri et al., 1996).
Proteases, however, are not easy to assay with their naturally occurring substrates. Moreover, many currently available synthetic substrates are expensive, insensitive, and nonselective. Furthermore, the use of high concentrations of the target protease, with either the naturally occurring substrate or a synthetic substrate, may be required for the assay, which may result in the self destruction of the protease.
Numerous chromogenic and fluorogenic substrates have been used to measure proteases (Monsees et al., 1994; Monsees et al., 1995) and modified luciferins have provided alternatives to fluorescent indicators (U.S. Pat. Nos. 5,035,999 and 5,098,828). Methods for using modified luciferins with a recognition site for a hydrolase as a pro-substrate were first described by Miska and Geiger (1989). These heterogenous assays were conducted by incubating the modified luciferin with a hydrolase for a specified period of time, then transferring an aliquot of the mixture to a solution containing luciferase. Masuda-Nishimura et al. (2000) reported the use of a single tube (homogenous) assay which employed a galactosidase substrate-modified luciferin. A non-heterogeneous luminescent protease assay has not yet been shown.
While luminescent assays are commonly known for their sensitivity, their performance relative to fluorescent assays is difficult to predict due to fundamental differences in assay formats. Specifically, enzyme-linked luminescence assays yield light coupled to the instantaneous rate of catalysis. In contrast, enzyme-linked fluorescence assays yield light based on the cumulative catalytic activity measured over a period of time (a so-called “endpoint” assay based upon accumulation of fluorophore). By integrating the catalytic activity over a period of time that can extend from hours to days, the light signal from a fluorescent assay can be greatly increased. Similar integration over such long periods is not practical for luminescent assays.
Thus, what is needed is a method to monitor protease activity that is a rapid, single-tube, homogeneous, sensitive assay.